ABCC1 p.Pro196Ala
| Predicted by SNAP2: | A: D (53%), C: N (61%), D: D (63%), E: D (66%), F: D (59%), G: D (59%), H: N (53%), I: D (53%), K: D (63%), L: D (63%), M: D (53%), N: N (53%), Q: N (53%), R: D (63%), S: N (57%), T: D (53%), V: D (63%), W: D (66%), Y: D (59%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Mutation of a single conserved tryptophan in multi... J Biol Chem. 2001 May 11;276(19):15616-24. Epub 2001 Feb 21. Ito K, Olsen SL, Qiu W, Deeley RG, Cole SP
Mutation of a single conserved tryptophan in multidrug resistance protein 1 (MRP1/ABCC1) results in loss of drug resistance and selective loss of organic anion transport.
J Biol Chem. 2001 May 11;276(19):15616-24. Epub 2001 Feb 21., 2001-05-11 [PMID:11278867]
Abstract [show]
Multidrug resistance protein 1 (MRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily and is capable of conferring resistance to a broad range of chemotherapeutic agents and transporting structurally diverse conjugated organic anions. In this study, we found that substitution of a highly conserved tryptophan at position 1246 with cysteine (W1246C-MRP1) in the putative last transmembrane segment (TM17) of MRP1 eliminated 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport by membrane vesicles prepared from transiently transfected human embryonic kidney cells while leaving the capacity for leukotriene C(4)- and verapamil-stimulated glutathione transport intact. In addition, in contrast to wild-type MRP1, leukotriene C(4) transport by the W1246C-MRP1 protein was no longer inhibitable by E(2)17betaG, indicating that the mutant protein had lost the ability to bind the glucuronide. A similar phenotype was observed when Trp(1246) was replaced with Ala, Phe, and Tyr. Confocal microscopy of cells expressing Trp(1246) mutant MRP1 molecules fused at the C terminus with green fluorescent protein showed that they were correctly routed to the plasma membrane. In addition to the loss of E(2)17betaG transport, HeLa cells stably transfected with W1246C-MRP1 cDNA were not resistant to the Vinca alkaloid vincristine and accumulated levels of [(3)H]vincristine comparable to those in vector control-transfected cells. Cells expressing W1246C-MRP1 were also not resistant to cationic anthracyclines (doxorubicin, daunorubicin) or the electroneutral epipodophyllotoxin VP-16. In contrast, resistance to sodium arsenite was only partially diminished, and resistance to potassium antimony tartrate remained comparable to that of cells expressing wild-type MRP1. This suggests that the structural determinants required for transport of heavy metal oxyanions differ from those for chemotherapeutic agents. Our results provide the first example of a tryptophan residue being so critically important for substrate specificity in a eukaryotic ATP-binding cassette transporter.
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No. Sentence Comment
103 RESULTS Identification of Trp1246 as a Functionally Important Amino Acid in MRP1-During the course of a mutational analysis of proline residues in MRP1, we generated an MRP1 mutant in which proline at position 196 was replaced with alanine (P196A).
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ABCC1 p.Pro196Ala 11278867:103:241
status: NEW105 In contrast, a second independently derived P196A mutant HeLa cell line displayed a phenotype similar to that of cells expressing wild-type MRP1.2 Using three sets of overlapping primers, the integrated MRP1-transfected cDNAs of the wild-type MRP1 and the first P196A mutant cell lines were amplified by polymerase chain reaction.
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ABCC1 p.Pro196Ala 11278867:105:44
status: NEWX
ABCC1 p.Pro196Ala 11278867:105:262
status: NEW106 The DNA fragments obtained were of the size expected, and when sequenced, it was discovered that, in addition to the P196A mutation, the transfected copies of MRP1 cDNA in the first cell mutant line with an altered phenotype contained a G 3 C nucleotide mutation, resulting in substitution of cysteine for tryptophan at position 1246.
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ABCC1 p.Pro196Ala 11278867:106:117
status: NEW191 In the course of a mutational analysis of this region of MRP1, we derived a mutant in which Pro196 was replaced with Ala by site-directed mutagenesis (34).
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ABCC1 p.Pro196Ala 11278867:191:92
status: NEW205 In contrast, a second independently derived transfected cell line also expressing an MRP1 protein with the same P196A mutation displayed a phenotype similar to that of cells expressing wild-type MRP1.2 It was subsequently discovered that the transfected MRP1 in the first P196A mutant cell line had acquired a second non-engineered mutation causing the substitution of Cys for Trp at position 1246.
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ABCC1 p.Pro196Ala 11278867:205:112
status: NEWX
ABCC1 p.Pro196Ala 11278867:205:272
status: NEW206 Consequently, we generated a single W1246C mutant of MRP1 so that its phenotype could be compared with that of the double P196A/W1246C mutant.
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ABCC1 p.Pro196Ala 11278867:206:122
status: NEW209 However, as observed for the double P196A/ W1246C mutant, transport of E217betaG by W1246C-MRP1 membrane vesicles was not detectably different from that obtained with vesicles from vector control-transfected cells, and although LTC4 transport appeared unchanged in the W1246C mutants, it could no longer be inhibited by E217betaG.
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ABCC1 p.Pro196Ala 11278867:209:36
status: NEW210 Taken together, these results allowed us to conclude that the altered phenotype of the P196A/W1246C-transfected cells was attributable to the single tryptophan substitution at position 1246 in MSD3 and did not require the additional proline substitution at position 196 in the cytoplasmic loop between MSD1 and MSD2.
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ABCC1 p.Pro196Ala 11278867:210:87
status: NEW[hide] Mutation of proline residues in the NH(2)-terminal... Biochim Biophys Acta. 2003 Sep 2;1615(1-2):103-14. Ito K, Weigl KE, Deeley RG, Cole SP
Mutation of proline residues in the NH(2)-terminal region of the multidrug resistance protein, MRP1 (ABCC1): effects on protein expression, membrane localization, and transport function.
Biochim Biophys Acta. 2003 Sep 2;1615(1-2):103-14., [PMID:12948592]
Abstract [show]
The Multidrug Resistance Protein, MRP1 (ABCC1) confers drug resistance and transports organic anions such as leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG). Previous studies showed that portions of the first membrane spanning domain (MSD1) and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. We have replaced 12 prolines in MSD1 and CL3 with alanine and determined the effects of these substitutions on MRP1 expression and transport activity. All singly substituted MRP1-Pro mutants could be expressed in HeLa cells, except MRP1-P104A. The expressed mutants also transported LTC(4) and E(2)17betaG, and their K(m) (LTC(4)) values were similar to wild-type MRP1. Expression of the double mutant MRP1-P42/51A was reduced by >80% although it localized to the plasma membrane and transported organic anions. MRP1 expression was also reduced when the first transmembrane helix (amino acids 37-54) was deleted. In contrast, the phenotypes of the multiply substituted CL3 mutants MRP1-P196/205/207/209A and MRP1-P235/255A were comparable to wild-type MRP1. However, Pro(255)-substituted MRP1 mutants showed reduced immunoreactivity with a monoclonal antibody (MAb) whose epitope is located in CL3. We conclude that certain prolines in MSD1 and CL3 play a role in the expression and structure of MRP1.
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No. Sentence Comment
47 For substitution of Pro residues in the CL3 loop connecting MSD1 to MSD2, the mutagenic primers were: MRP1-P196A (5V-CA GAT CGC TCA GCC CTG TTC TC-3V), MRP1-P205A (5V-C ATC CAC GAC GCT AAT CCC TG-3V), MRP1-P207A (5V-C GAC CCT AAT GCC TGC CCA GAG-3V), MRP1-P209A (5V-CTA ATC CCT GCG CAG AGT CCA G-3V), MRP1-P235A (5V-C TAC CGC CAG GCC CTG GAG GG-3V), MRP1-P255A (5V-CG GAA CAA GTC GTG GCT GTT TTG G-3V), MRP1-P272A (5V-CT AGG AAG CAG GCG GTG AAG G-3V).
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ABCC1 p.Pro196Ala 12948592:47:107
status: NEW49 The quadruple MRP1-P196/205/207/209A construct was generated by introducing the P205/207/209A mutations into the MRP1-P196A construct.
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ABCC1 p.Pro196Ala 12948592:49:118
status: NEW107 The seven single (P196A, P205A, P207A, P209A, P235A, P255A, and P272A) and two multiply substituted CL3 mutants (P196/205/207/209A and P235/255A) could also be stably expressed in HeLa cells.
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ABCC1 p.Pro196Ala 12948592:107:18
status: NEW188 Results were expressed relative to wild-type Table 1 Kinetic parameters of [3 H]LTC4 uptake by membrane vesicles prepared from transfected HeLa cells expressing MRP1-Pro mutants Transfected cell line Km (nM) Vmax (pmol minÀ 1 mgÀ 1 ) Normalized Vmax MSD1 mutants WT-MRP1 128 80 80 P42A 139 35 41 P51A 109 55 50 P42/51A 103 15 48 P69A 142 46 42 P110A 150 73 124 CL3 mutants WT-MRP1 88 57 57 P196A 143 25 52 P205A 63 14 88 P207A 81 75 110 P209A 112 121 85 P196/205/207/209A 90 87 94 P235A 91 45 30 P255A 99 110 67 P235/255A 97 81 35 P272A 80 59 109 The kinetic parameters of [3 H]LTC4 uptake were determined by regression analysis of the Eadie-Hofstee transformed data.
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ABCC1 p.Pro196Ala 12948592:188:400
status: NEW187 Results were expressed relative to wild-type Table 1 Kinetic parameters of [3 H]LTC4 uptake by membrane vesicles prepared from transfected HeLa cells expressing MRP1-Pro mutants Transfected cell line Km (nM) Vmax (pmol min 1 mg 1 ) Normalized Vmax MSD1 mutants WT-MRP1 128 80 80 P42A 139 35 41 P51A 109 55 50 P42/51A 103 15 48 P69A 142 46 42 P110A 150 73 124 CL3 mutants WT-MRP1 88 57 57 P196A 143 25 52 P205A 63 14 88 P207A 81 75 110 P209A 112 121 85 P196/205/207/209A 90 87 94 P235A 91 45 30 P255A 99 110 67 P235/255A 97 81 35 P272A 80 59 109 The kinetic parameters of [3 H]LTC4 uptake were determined by regression analysis of the Eadie-Hofstee transformed data.
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ABCC1 p.Pro196Ala 12948592:187:388
status: NEW